A novel approach has been implemented to study the activation state of protein kinases en masse. Our methods allow isolation and analysis of protein kinases from cell lines, tissues and tumors assayed in a single mass spectrometry run using Multiplexed Inhibitor Beads (MIBs). MIBs consist of mixtures of Sepharose beads with covalently immobilized, linker adapted, kinase inhibitors. Kinase capture is reproducible and is a function of affinity of kinases for the different immobilized inhibitors as well as the activation state of the kinase. The inhibitors we have synthesized for linker conjugation to Sepharose beads are primarily type 1 inhibitors that preferentially bind activated states of protein kinases. Extensive characterization of the MIBs has demonstrated they have a strong preference for binding the active state of kinases and inactive kinases bind poorly. Using a layered bead composition for MIB columns, we are able to capture and identify more than 275 kinases in a single mass spectrometry run. Based on RNA-seq analysis, MIBs are capturing at least 75-80% of the expressed kinome from cell lines, tumors or normal tissues. Importantly, we have captured and characterized up to 275 kinases with as little as 500 mg protein from cell and tissue lysates. This level of sensitivity is essential for kinome analysis when tissue is limiting. Thus, MIB/MS provides an unparalleled scalable biology- based high throughput technology to assay the activation state of the kinome including untargeted and understudied kinases. There simply are few or no reagents for many of the understudied kinases, thereby limiting their study and MIB/MS overcomes these limitations. MIB/MS also detects activity changes in kinases for which reagents cannot distinguish closely related kinases (e.g., different activity states of MEK1 vs. MEK2 and ERK1 vs. ERK2, which are assessed together by anti-phospho-antibodies that cannot distinguish isoforms). Similarly, we can easily distinguish the regulation of related receptor tyrosine kinases (RTKs), such as DDR1 and DDR2, for which there are few reagents for study.
The aims will define comprehensive kinome activation signatures that include poorly or uncharacterized kinases. Aims i nclude: 1. Determine the activation state of the kinome using MIB/MS for a series of human cell lines spanning different cancer types, human and mouse tumors, and normal tissues of the mouse. 2. Determine the response of the kinome to perturbation of cellular physiology using specific chemical probes targeting protein and lipid kinases, cytoskeleton, metabolic regulators, DNA damage and epigenetic chromatin modifying enzymes. 3. Determine the consequence of RNAi knockdown of understudied kinases on the activation state of the kinome.
The aims emphasize determining the activation state of understudied kinases and if specific understudied kinases function as part of a signature of kinase activation/inhibition in response to specific cellular perturbations. This analysis will defne the signaling networks and cellular functions for which the activity of understudied kinases are regulated and contribute to homeostatic control mechanisms. The goal is to identify and validate which current understudied kinases function as regulatory nodes within the kinome and warrant future chemical probe development.